Cardiac fibroblasts (CFs), the most abundant nonmyocyte cell type in the heart, provide continuity between cardiomyocytes, maintain the structural integrity of the myocardium and coronary vasculature and are the predominant cellular mediators of fibrosis in the heart in response to injury. Recent efforts to induce lineage conversion of CFs to cardiomyocytes and endothelial cells, in an effort to generate new contractile tissue and blood vessels, have been limited by an inefficient reprogramming. A robust and efficient method involves precise stoichiometry as well as certain levels of expression of reprogramming factors in a temporally defined sequence. Additionally, a fundamental obstacle remains the inability to identify a homogenous population of fibroblasts susceptible to reprogramming. The central hypothesis of this proposal is that a discrete population of CFs that share the same embryonic origin as cardiomyocytes or endothelial cells may have an epigenetic 'memory' that predisposes them to direct reprogramming following a dose-titratable, temporally-controlled, and stage-specific sequential delivery of required transcription factors (TFs). By using modified RNA (modRNA) as a non-integrating genetic delivery system, we can achieve a controlled 'pulse-like' expression of core TFs and avoid potential issues associated with viral integration. To address our hypotheses, we will use lineage-tracing experiments to identify discrete populations of CFs that share a common ancestor with cardiomyocytes and/or with endothelial cells. Synthetic modRNAs that encode core-reprogramming TFs will be used for lineage conversion both in vitro and in vivo. We believe that the native milieu of the intact heart, surrounding contractile cells, growth factors ad cytokines will provide a more permissive environment for functional reprogramming. Therefore, it is expected that delivery of modRNA to the injured area of the heart where there is active and dynamic proliferation of fibroblasts could result in an efficient reprogramming of CFs to cardiomyocytes and endothelial cells. The challenges associated with this project are substantial; however, its potential scientific and clinical impact is significant. We are confident that our meticulous experimental design and innovative approach will unravel the origin of CFs and their contribution to cardiac development and injury. Additionally, our findings will open entirely new avenues to manipulate CFs in vivo using modRNAs to restore damaged myocardium and reverse fibrosis.